Dual stress factors adaptive evolution for high EPA production in Schizochytrium sp. and metabolomics mechanism analysis.

Eicosapentaenoic acid (EPA) is a vital É·-3 polyunsaturated fatty acid (PUFA) for human body with various physiological functions. In this study, we proposed an adaptive evolutionary strategy based on high-temperature and high-oxygen two-factor stress to increase the EPA production capacity of Schizochytrium. High-temperature stress was used to increase EPA yield, and high oxygen was implemented to continuously stimulate cell growth and lipid accumulation. The biomass and EPA production of ALE-D50 reached 35.33 g/L and 1.54 g/L, which were 43.85% and 71.11% higher than that of the original strain, respectively. Lower in vivo reactive oxygen species levels indicated that the evolved strain possessed stronger antioxidant activity. Liquid chromatography-mass spectrometry metabolomics showed that enhanced glucose consumption and glycolysis metabolism, as well as a weakened tricarboxylic acid cycle and reduced amino acid metabolic tributaries in the evolved strain, might be associated with increased growth and EPA synthesis. Finally, the lipid production and EPA production in a fed-batch fermentation were further increased to 48.93 g/L and 3.55 g/L, improving by 54.30% and 90.86%, respectively. This study provides a novel pathway for promoting EPA biosynthesis in Schizochytrium.

Production of docosahexaenoic acid by a novel isolated Aurantiochytrium sp. 6-2 using fermented defatted soybean as a nitrogen source for sustainable fish feed development.

We focused on the production of docosahexaenoic acid (DHA)-containing microbial lipids by Aurantiochytrium sp. using of defatted soybean (DS) as a nitrogen source. Defatted soybean is a plant biomass that could provide a sustainable supply at a low cost. Results showed that Aurantiochytrium sp. could not directly assimilate the DS as a nitrogen source but could grow well in a medium containing DS fermented with rice malt. When cultivated in a fermented DS (FDS) medium, Aurantiochytrium sp. showed vigorous growth with the addition of sufficient sulfate and chloride ions as inorganic nutrients without seawater salt. A novel isolated Aurantiochytrium sp. 6-2 showed 15.8 ± 3.4 g/L DHA productivity (in 54.8 ± 12.1 g/L total fatty acid production) in 1 L of the FDS medium. Therefore, DHA produced by Aurantiochytrium sp. using FDS enables a stable and sustainable DHA supply and could be an alternative source of natural DHA derived from fish oil.

Accumulation of docosapentaenoic acid (n-3 DPA) in a novel isolate of the marine ichthyosporean Sphaeroforma arctica.

OBJECTIVE: Currently, there is lack of a consistent and highly enriched source for docosapentaenoic acid (n-3 DPA, C22:5), and this work report the isolation of microorganism that naturally produces n-3 DPA. RESULTS: In this work, we screened microorganisms in our culture collections with the goal to isolate a strain with high levels of n-3 DPA. We isolated a strain of Sphaeroforma arctica that produces up to 11% n-3 DPA in total fatty acid and has a high n-3 DPA to DHA/EPA ratio. The cell growth of the isolated strain was characterized using microscopy imaging and flow cytometer technologies to confirm the coenocytic pattern of cell divisions previously described in S. arctica. Our novel isolate of S. arctica grew more robustly and produced significantly more n-3 DPA compared to previously isolated and described strains indicating the uniqueness of the discovered strain. CONCLUSION: Overall, this work reports a first isolate n-3 DPA producing microorganism and establishes the foundation for future strain improvement and elucidation of the physiological function of this LC-PUFA for human nutrition and health.

Concentration-dependent dual roles of proanthocyanidins on oxidative stress and docosahexaenoic acid production in Schizochytrium sp. ATCC 20888.

Antioxidant addition is an effective strategy to achieve docosahexaenoic acid (DHA) overproduction in oleaginous microorganisms. Nevertheless, antioxidants like phenolic compounds sometimes exert pro-oxidant activity. In this work, effects of proanthocyanidins (PAs) on fermentation performance and oxidative stress in Schizochytrium sp. were investigated. Low PAs addition (5 mg/L) reduced reactive oxygen species and enhanced lipogenic enzymes activities and NADPH, resulting in significant increase in lipid (20.3 g/L) by 33.6 % and DHA yield (9.8 g/L) by 53.4 %. In contrast, high PAs addition (500 mg/L) exerted pro-oxidant effects, aggravated oxidative damage and lipid peroxidation, leading to sharp decrease in biomass (21.3 g/L) by 35.1 %, lipid (8.2 g/L) by 46.0 %, and DHA (2.9 g/L) by 54.8 %. Therefore, the antioxidant concentration is especially crucial in DHA production. This study is the first to report concentration-dependant dual roles of PAs in oxidative stress and DHA production in Schizochytrium sp., providing new insights into microbial DHA production.

Integration analysis of ATAC-seq and RNA-seq provides insight into fatty acid biosynthesis in Schizochytrium limacinum under nitrogen limitation stress.

BACKGROUND: Schizochytrium limacinum holds significant value utilized in the industrial-scale synthesis of natural DHA. Nitrogen-limited treatment can effectively increase the content of fatty acids and DHA, but there is currently no research on chromatin accessibility during the process of transcript regulation. The objective of this research was to delve into the workings of fatty acid production in S. limacinum by examining the accessibility of promoters and profiling gene expressions. RESULTS: Results showed that differentially accessible chromatin regions (DARs)-associated genes were enriched in fatty acid metabolism, signal transduction mechanisms, and energy production. By identifying and annotating DARs-associated motifs, the study obtained 54 target transcription factor classes, including BPC, RAMOSA1, SPI1, MYC, and MYB families. Transcriptomics results revealed that several differentially expressed genes (DEGs), including SlFAD2, SlALDH, SlCAS1, SlNSDHL, and SlDGKI, are directly related to the biosynthesis of fatty acids, meanwhile, SlRPS6KA, SlCAMK1, SlMYB3R1, and SlMYB3R5 serve as transcription factors that could potentially influence the regulation of fatty acid production. In the integration analysis of DARs and ATAC-seq, 13 genes were identified, which were shared by both DEGs and DARs-associated genes, including SlCAKM, SlRP2, SlSHOC2, SlTN, SlSGK2, SlHMP, SlOGT, SlclpB, and SlDNAAF3. CONCLUSIONS: SlCAKM may act as a negative regulator of fatty acid and DHA synthesis, while SlSGK2 may act as a positive regulator, which requires further study in the future. These insights enhance our comprehension of the processes underlying fatty acid and DHA production in S. limacinum. They also supply a foundational theoretical framework and practical assistance for the development of strains rich in fatty acids and DHA.

Enhancing docosahexaenoic acid production in Aurantiochytrium species using atmospheric and room temperature plasma mutagenesis and comprehensive multi-omics analysis.

Aurantiochytrium sp. belongs to marine heterotrophic single-cell protist, which is an important decomposer in marine ecosystem. Aurantiochytrium sp. has gained notoriety because of its ability to accumulate high-value docosahexaenoic acid (DHA), but the key factors of DHA synthesis were unclear at present. In this study, Atmospheric and Room Temperature Plasma technology was applied to the mutagenic breeding of Aurantiochytrium sp., and transcriptomics and proteomics were adopted to analyze the DHA-biosynthesis mechanism. According to the growth and DHA accumulation profiles, the mutant strain Aurantiochytrium sp. R2A35 was selected. The DHA content in total lipids was greatly improved from 49.39 % of the wild strain R2 to 63.69 % of the mutant strain. Moreover, the DHA content in the biomass of Aurantiochytrium sp. R2A35 as 39.72 % was the highest DHA productivity reported so far. The differentially expressed genes distinguished from transcriptome and the TMT-identified differential proteins distinguished from proteome confirmed that the expression of acetyl-CoA carboxylase and ketoacyl reductase was up-regulated by 4.78-fold and 6.95-fold, respectively and the fatty acid synthase was concurrently down-regulated by 2.79-fold, so that more precursor was transported to the polyketide synthase pathway, thereby increasing the DHA yield in Aurantiochytrium sp. R2A35. This research would provide reference for the DHA metabolism process and contribute to the understanding of the decomposer - Aurantiochytrium sp. in marine ecosystems.

bZIP transcription factor FabR: Redox-dependent mechanism controlling docosahexaenoic acid biosynthesis and H2O2 stress response in Schizochytrium sp.

Schizochytrium sp. is an important industrial strain for commercial production of docosahexaenoic acid (DHA), which plays essential physiological roles in infant development and human health. The regulatory network for DHA biosynthesis and lipid accumulation in Schizochytrium remains poorly understood. FabR (fatty acid biosynthesis repressor), a basic leucine zipper (bZIP) transcription factor, was transcriptionally downregulated under low-nitrogen condition. Deletion of fabR gene (mutant ΔfabR) increased production of total lipids and DHA by 30.1% and 46.5%, respectively. ΔfabR displayed H2O2 stress resistance higher than that of parental strain or complementation strain CfabR. FabR bound specifically to 7-bp pseudo-palindromic sequence 5'-ATTSAAT-3' in upstream regions and repressed transcription of fatty acid biosynthesis genes (acl, fas, pfa) and antioxidant defense genes (cat, sod1, sod2, gpx). DNA binding activity of FabR was regulated in a redox-dependent manner. Under oxidative condition, FabR forms intermolecular disulfide bonds between two Cys46 residues of dimers; its DNA binding activity is thereby lost, and the transcription of its target genes is enhanced through derepression. Our findings clarify the redox-dependent mechanism that modulates FabR activity governing lipid and DHA biosynthesis and H2O2 stress response in Schizochytrium.

A novel sulfur supply strategy for maximizing lipid production in Tribonema minus (Xanthophyceae).

Tribonema minus, a promising filamentous oleaginous microalga, was cultured under different nutrient concentrations and different culture modes (fed-batch culture, two-step culture) to study the method of rapid regulation of its lipid metabolism. In contrast to many other oleaginous microalgae, T. minus did not show that nitrogen stress promoted lipid accumulation; however, sulfur deficiency promoted rapid lipid accumulation with a maximum lipid content of 54% of dry weight. Increasing the MgSO4 concentration significantly increased nitrogen uptake and biomass (10.09 g/L). Lipid productivity was significantly increased by the two-step culture using a medium with a high concentration of MgSO4 in the first step and a sulfur-free medium in the second step. In addition, it was found that the lipid content of T. minus was negatively correlated with the intracellular sulfur content when the intracellular sulfur content was below 0.6%. This study provides a new approach for industrial lipid production in T. minus.

Uncovering global lipid accumulation routes towards docosahexaenoic acid (DHA) production in Aurantiochytrium sp. SW1 using integrative proteomic analysis.

Aurantiochytrium sp., a marine thraustochytrid possesses a remarkable ability to produce lipid rich in polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA). Although gene regulation underlying lipid biosynthesis has been previously reported, proteomic analysis is still limited. In this study, high DHA accumulating strain Aurantiochytrium sp. SW1 has been used as a study model to elucidate the alteration in proteome profile under different cultivation phases i.e. growth, nitrogen-limitation and lipid accumulation. Of the total of 5146 identified proteins, 852 proteins were differentially expressed proteins (DEPs). The largest number of DEPs (488 proteins) was found to be uniquely expressed between lipid accumulating phase and growth phase. Interestingly, there were up-regulated proteins involved in glycolysis, glycerolipid, carotenoid and glutathione metabolism which were preferable metabolic routes towards lipid accumulation and DHA production as well as cellular oxidative defence. Integrated proteomic and transcriptomic data were also conducted to comprehend the gene and protein regulation underlying the lipid and DHA biosynthesis. A significant up-regulation of acetyl-CoA synthetase was observed which suggests alternative route of acetate metabolism for acetyl-CoA producer. This study presents the holistic routes underlying lipid accumulation and DHA production in Aurantiochytrium sp. SW1 and other relevant thraustochytrid.

Enhancing the accumulation of lipid and docosahexaenoic acid in Schizochytrium sp. by co-overexpression of phosphopantetheinyl transferase and ω-3 fatty acid desaturase.

Docosahexaenoic acid (DHA) as one of ω-3 polyunsaturated fatty acids (PUFAs), plays a key role in brain development, and is widely used in food additives and the pharmaceutical industry. Schizochytrium sp. is often considered as a satisfactory strain for DHA industrialization. The aim of this study was to assess the feasibility of phosphopantetheinyl transferase (PPTase) and ω-3 fatty acid desaturase (FAD) for regulating DHA content in Schizochytrium sp. PPTase is essential to activate the polyketide-like synthase (PKS) pathway, which can transfer apo-acyl-carrier protein (apo-ACP) into holo-ACP, and plays a key role in DHA synthesis. Moreover, DHA and docosapentaenoic acid (DPA) are synthesized by the PKS pathway simultaneously, so high DPA synthesis limits the increase of DHA content. In addition, the detailed mechanisms of PKS pathway have not been fully elucidated, so it is difficult to improve DHA content by modifying PKS. However, ω-3 FAD can convert DPA into DHA, and it is the most direct and effective way to increase DHA content and reduce DPA content. Based on this, PPTase was overexpressed to enhance the synthesis of DHA by the PKS pathway, overexpressed ω-3 FAD to convert the co-product of the PKS pathway into DHA, and co-overexpressed PPTase and ω-3 FAD. With these strategies, compared with wild type, the final lipid, and DHA titer were 92.5 and 51.5 g L-1 , which increased by 46.4% and 78.1%, respectively. This study established an efficient DHA production strain, and provided some feasible strategies for industrial DHA production in Schizochytrium sp.

Overexpression of S-R enhances the accumulation of biomass, fatty acids, and ß-carotene in Schizochytrium.

Strategies for enhancing biomass accumulation and increasing the production of fatty acids and ß-carotene in Schizochytrium are hindered by the lack of suitable targets. In this study, S-R, a RING (really interesting new gene) finger domain-containing protein, was identified in Schizochytrium, with homologs found in the family Thraustochytriaceae. Transgenic strains overexpressing S-R showed a minor improvement in cell growth but a significant increase in total fatty acids content by 1.29- to 1.36-fold. Almost all individual saturated fatty acids exhibited significant increases, with the greatest increase observed in the C14:0 content, by 1.52- to 1.78-fold. Additionally, the ß-carotene content of S-R strains was significantly upregulated. Overexpression of s-r conferred hypersaline tolerance in Schizochytrium, with a significant increase in dry cell weight, total fatty acids and ß-carotene, likely due to the upregulation of glycerol and proline. This study provides a feasible strategy to engineer Thraustochytriaceae for efficient biomass and biochemical production.

Prevention of UVB Induced Metabolic Changes in Epidermal Cells by Lipid Extract from Microalgae Nannochloropsis oceanica.

The exposure of skin cells to UV radiation leads to redox imbalances and inflammation. The present study investigates a lipid extract obtained from the microalga Nannochloropsis oceanica as a potential protector against UVB-induced disturbances in human keratinocytes. The findings of this study show that the Nannochloropsis oceanica extract significantly inhibits UVB-induced cell death while concurrently decreasing the activity of pro-oxidative enzymes (xanthine and NADPH oxidase) and reducing the levels of ROS. Furthermore, the extract augments the activity of antioxidant enzymes (superoxide dismutases and catalase), as well as glutathione/thioredoxin-dependent systems in UVB-irradiated cells. The expression of Nrf2 factor activators (p62, KAP1, p38) was significantly elevated, while no impact was observed on Nrf2 inhibitors (Keap1, Bach1). The antioxidant activity of the extract was accompanied by the silencing of overexpressed membrane transporters caused by UVB radiation. Furthermore, the Nannochloropsis oceanica extract exhibited anti-inflammatory effects in UVB-irradiated keratinocytes by decreasing the levels of TNFα, 8-iso prostaglandin F2, and 4-HNE-protein adducts. In conclusion, the lipid components of Nannochloropsis oceanica extract effectively prevent the pro-oxidative and pro-inflammatory effects of UVB radiation in keratinocytes, thereby stabilizing the natural metabolism of skin cells.

Bioprocess conditions and regulation factors to optimize squalene production in thraustochytrids.

Squalene is a widely distributed natural triterpene, as it is a key precursor in the biosynthesis of all sterols. It is a compound of high commercial value worldwide because it has nutritional, medicinal, pharmaceutical, and cosmetic applications, due to its different biological properties. The main source of extraction has been shark liver oil, which is currently unviable on a larger scale due to the impacts of overexploitation. Secondary sources are mainly vegetable oils, although a limited one, as they allow low productive yields. Due to the diversity of applications that squalene presents and its growing demand, there is an increasing interest in identifying sustainable sources of extraction. Wild species of thraustochytrids, which are heterotrophic protists, have been identified to have the highest squalene content compared to bacteria, yeasts, microalgae, and vegetable sources. Several studies have been carried out to identify the bioprocess conditions and regulation factors, such as the use of eustressors that promote an increase in the production of this triterpene; however, studies focused on optimizing their productive yields are still in its infancy. This review includes the current trends that also comprises the advances in genetic regulations in these microorganisms, with a view to identify the culture conditions that have been favorable in increasing the production of squalene, and the influences that both bioprocess conditions and applied regulation factors partake at a metabolic level.

Coupling and decoupling of marine stramenopiles and cyanobacteria in eutrophic coastal waters of Korea.

The spatiotemporal distribution of MASTs (MArine STramenopiles), mostly affiliated with heterotrophic protists, and their interactions with Synechococcales were investigated in an anthropogenically polluted bay of the East Sea using 18S rRNA and 16S rRNA gene sequences. The bay was characterized by strong stratification between the surface and bottom layers and cold and nutrient-rich water intrusion in summer, whereas the bay water was well mixed in winter. MAST-3, MAST-6, MAST-7, and MAST-9 were the major MAST clades, whereas the dominance of MAST-9 declined from >80 % in summer to <10 % in winter and the diversity of MAST communities increased in winter. Co-occurrence network analysis via the sparse partial least squares revealed that MAST-3 had a Synechococcales-specific interaction during the study periods but prey-specific interactions with other MAST clades were not detected. Temperature and salinity markedly influenced the relative abundance of major MAST clades. The relative abundance of MAST-3 increased at temperatures above 20 °C and salinities above 33 ‰, however, the abundance of MAST-9 decreased under the same conditions. Analysis of the metabolic functions of cyanobacteria using the FAPROTAX (Functional Annotation of Prokaryotic Taxa) indicated that the response of photosynthetic cyanobacteria to NH4+ and PO43- was significant in summer but these functions were not tightly coupled with the abundance of Synechococcales. Similarly, strong associations of MAST-3 with high temperature/salinity and Synechococcales were indicative of coupled cascading during bottom-up processes. However, other major MAST clades were likely decoupled with Synechococcales and environmental conditions in which cyanobacteria can thrive. Therefore, our results demonstrated that MAST communities can couple or decouple with environmental variables and potential prey depending on MAST clades. Collectively, our findings provide novel insights into the role of MAST communities in microbial food webs in eutrophic coastal waters.

Effects of sodium selenite on the growth, biochemical composition and selenium biotransformation of the filamentous microalga Tribonema minus.

This study aimed to investigate the physiological and biochemical responses of filamentous microalga Tribonema minus to different Na2SeO3 concentrations and its selenium absorption and metabolism to evaluate the potential in treating selenium-containing wastewater. The results showed that low Na2SeO3 concentrations promoted growth by increasing chlorophyll content and antioxidant capacity, whereas high concentrations caused oxidative damage. Although Na2SeO3 exposure reduced lipid accumulation compared with the control, it significantly increased carbohydrate, soluble sugar, and protein contents, with the highest carbohydrate productivity of 117.97 mg/L/d at 0.5 mg/L Na2SeO3. Furthermore, this alga effectively absorbed Na2SeO3 in the growth medium and converted most of it into volatile selenium and a small part into organic selenium (predominantly as selenocysteine), showing strong selenite removal efficacy. This is the first report on the potential of T. minus to produce valuable biomass while removing selenite, providing new insights into the economic feasibility of bioremediation of selenium-containing wastewater.

Extraction of Nannochloropsis Fatty Acids Using Different Green Technologies: The Current Path.

Nannochloropsis is a genus of microalgae widely recognized as potential sources of distinct lipids, particularly polyunsaturated fatty acids (PUFA). These may be obtained through extraction, which has conventionally been performed using hazardous organic solvents. To substitute such solvents with "greener" alternatives, several technologies have been studied to increase their extraction potential. Distinct technologies utilize different principles to achieve such objective; while some aim at disrupting the cell walls of the microalgae, others target the extraction per se. While some methods have been utilized independently, several technologies have also been combined, which has proven to be an effective strategy. The current review focuses on the technologies explored in the last five years to extract or increase extraction yields of fatty acids from Nannochloropsis microalgae. Depending on the extraction efficacy of the different technologies, distinct types of lipids and/or fatty acids are obtained accordingly. Moreover, the extraction efficiency may vary depending on the Nannochloropsis species. Hence, a case-by-case assessment must be conducted in order to ascertain the most suited technology, or tailor a specific one, to be applied to recover a particular fatty acid (or fatty acid class), namely PUFA, including eicosapentaenoic acid.

Development of Schizochytrium sp. strain HS01 with high-DHA and low-saturated fatty acids production by multi-pronged adaptive evolution.

PURPOSE: Docosahexaenoic acid (DHA) is an important omega-3 unsaturated fatty acid and has been widely applied in medicine, food additives, and feed ingredients. The fermentative production of DHA using microorganisms, including Schizochytrium sp., attracted much attention due to its high production efficiency and environment friendly properties. An efficient laboratory evolution approach was used to improve the strain's performance in this study. METHODS: A multi-pronged laboratory evolution approach was applied to evolve high-yield DHA-producing Schizochytrium strain. We further employed comparative transcriptional analysis to identify transcriptional changes between the screened strain HS01 and its parent strain GS00. RESULTS: After multiple generations of ALE, a strain HS01 with higher DHA content and lower saturated fatty acids content was obtained. Low nitrogen conditions were important for enhancing DHA biosynthesis in HS01. The comparative transcriptional analysis results indicated that during the fermentation process of HS01, the expression of key enzymes in the glycolysis, the pentose phosphate pathway and the tricarboxylic acid cycle were up-regulated, while the expression of polyketide synthase genes and fatty acid synthesis genes were similar to those in GS00. CONCLUSION: The results suggest that the improved DHA production capacity of HS01 is not due to enhancement of the DHA biosynthesis pathway, but rather related to modulation of central metabolism pathways.

Enhancing the biomass and docosahexaenoic acid-rich lipid accumulation of Schizochytrium sp. in propionate wastewater.

In order to find a more effective way to obtain docosahexaenoic acid (DHA) rich lipid from Schizochytrium sp., a widespread propionate wastewater (PW) is used. PW is a common industrial and domestic wastewater, and transforming it into valuable products is a potential treatment method. Schizochytrium sp. is a rapidly growing oleaginous organism, which has been used commercially for DHA production. Herein, PW is completely used for DHA production by Schizochytrium sp. by genetic engineering and fermentation optimization, which can alleviate the increasingly tense demand for water resources and environmental pollution caused by industrial wastewater. Firstly, the methylmalonyl-CoA mutase (MCM) was overexpressed in Schizochytrium sp. to enhance the metabolism of propionate, then the engineered strain of overexpressed MCM (OMCM) can effectively use propionate. Then, the effects of PW with different concentration of propionate were investigated, and results showed that OMCM can completely replace clean water with PW containing 5 g L-1 propionate. Furthermore, in the fed-batch fermentation, the OMCM obtained the highest biomass of 113.4 g L-1 and lipid yield of 64.4 g L-1 in PW condition, which is 26.8% and 51.7% higher than that of wild type (WT) in PW condition. Moreover, to verify why overexpression of MCM can promote DHA and lipid accumulation, the comparative metabolomics, ATP production level, the antioxidant system, and the transcription of key genes were investigated. Results showed that ATP induced by PW condition could drive the synthesis of DHA, and remarkably improve the antioxidant capacity of cells by enhancing the carotenoids production. Therefore, PW can be used as an effective and economical substrate and water source for Schizochytrium sp. to accumulate biomass and DHA.

Functional metabolism pathways of significantly regulated genes in Nannochloropsis oceanica with various nitrogen/phosphorus nutrients for CO2 fixation.

To determine the optimal CO2 concentration for microalgal biomass cultivated with industrial flue gas and improve carbon fixation capacity and biomass production. Functional metabolism pathways of significantly regulated genes in Nannochloropsis oceanica (N. oceanica) with various nitrogen/phosphorus (N/P) nutrients for CO2 fixation were comprehensively clarified. At 100 % N/P nutrients, the optimum CO2 concentration was 70 % and the maximum biomass production of microalgae was 1.57 g/L. The optimum CO2 concentration was 50 % for N or P deficiency and 30 % for both N and P deficiency. The optimal combination of CO2 concentration and N/P nutrients caused significant up regulation of proteins related to photosynthesis and cellular respiration in the microalgae, enhancing photosynthetic electron transfer efficiency and carbon metabolism. Microalgal cells with P deficiency and optimal CO2 concentration expressed many phosphate transporter proteins to enhance P metabolism and N metabolism to maintain a high carbon fixation capacity. However, inappropriate combination of N/P nutrients and CO2 concentrations caused more errors in DNA replication and protein synthesis, generating more lysosomes and phagosomes. This inhibited carbon fixation and biomass production in the microalgae with increased cell apoptosis.

Transcriptome Analysis Reveals an Eicosapentaenoic Acid Accumulation Mechanism in a Schizochytrium sp. Mutant.

Eicosapentaenoic acid (EPA) is an omega-3 long-chain polyunsaturated fatty acid (PUFA) essential for human health. Schizochytrium is a marine eukaryote that has been widely utilized for the synthesis of PUFAs. The current low potency and performance of EPA production by fermentation of Schizochytrium spp. limits its prospect in commercial production of EPA. Since the synthesis pathway of EPA in Schizochytrium spp. is still unclear, mutagenesis combined with efficient screening methods are still desirable. In this study, a novel screening strategy was developed based on a two-step progressive mutagenesis method based on atmospheric and room temperature plasma (ARTP) and diethyl sulfate (DES) after multiple stresses (sethoxydim, triclosan and 2,2'-bipyridine) compound screening. Finally, the mutant strain DBT-64 with increased lipid (1.57-fold, 31.71 g/L) and EPA (5.64-fold, 1.86 g/L) production was screened from wild-type (W) strains; the docosahexaenoic acid (DHA) content of mutant DBT-64 (M) was 11.41% lower than that of wild-type strains. Comparative transcriptomic analysis showed that the expression of genes related to the polyketide synthase, fatty acid prolongation, and triglyceride synthesis pathways was significantly upregulated in the mutant strain, while the expression of genes involved in the ß-oxidation pathway and fatty acid degradation pathway was downregulated in favor of EPA biosynthesis in Schizochytrium. This study provides an effective strain improvement method to enhance EPA accumulation in Schizochytrium spp. IMPORTANCE Schizochytrium, a marine eukaryotic microorganism, has emerged as a candidate for the commercial production of PUFAs. EPA is an omega-3 PUFA with preventive and therapeutic effects against cardiovascular diseases, schizophrenia, and other disorders. Currently, the low potency and performance of EPA production by Schizochytrium spp. limits its commercialization. In this study, we performed two-step progressive mutagenesis based on ARTP and DES and screened multiple stresses (sethoxydim, triclosan, and 2,2'-bipyridine) to obtain the EPA-high-yielding Schizochytrium mutant. In addition, high expression of the polyketide synthase pathway, fatty acid elongation pathway, and triglyceride synthesis pathway in the mutants was confirmed by transcriptomic analysis. Therefore, the multistress screening platform established in this study is important for breeding EPA-producing Schizochytrium spp. and provides valuable information for regulating the proportion of EPA in microalgal lipids by means of genetic engineering.